1b. Approach (from AD-416)
We will work towards developing virus-resistant plants, using transgenic plant technology. Tomato spotted wilt virus (TSWV) has worldwide impact, and particularly in the tropics and subtropics. We have cloned and sequenced several TSWV genes, and transferred them to lettuce plants. We will evaluate these lines for TSWV resistance. In addition, we will evaluate lines of transgenic tomato for TSWV resistance. These tomato plants were developed by the PBARC research group, and contain a gene for TSWV resistance bred in using conventional means, in addition to a TSWV transgene. After initial greenhouse evaluations, we will move the most promising lines on to field trials. TSWV is an extremely important plant virus in Hawaii and elsewhere, and the development of TSWV-resistant lettuce and tomato will greatly benefit agriculture in Hawaii and abroad.
Work will also begin towards developing orchid plants with resistance to cymbidium mosaic virus (CymMV), the most important orchid virus worldwide. Most Hawaiian orchid growers that sell cut flowers specialize in a just a few Dendrobium varieties, and experience CymMV infection rates of over 90%. These growers have selected cultivars with good tolerance to virus infection, but they are now severely limited with regard to bringing in new cultivars, as well as selling potted plants. In addition, shipments of orchids from overseas are often infected with CymMV, so there is an influx of new CymMV strains to Hawaii. Recent techniques for engineering virus resistance in plants can provide resistance to multiple strains of a plant virus, and we have cloned and sequenced several of the Hawaiian isolates of CymMV. The availability of CymMV-resistant dendrobiums will benefit the orchid cut-flower growers directly, and will also aid the entire orchid industry by reducing the overall numbers of virus-infected plants.
Documents SCA with U of HI -Hilo.

3. Progress Report
The goal of this project is to develop virus-resistant plants using transgenic plant technology, which contributes directly to the objective of the in-house project.
Developing lettuce with resistance to tomato spotted wilt virus:
Plants with virus resistance can be developed using transgenic plant technology, by transferring a viral gene or gene fragment to a host plant. When successful, this results in crops with highly specific and heritable resistance. Tomato spotted wilt virus (TSWV) is the most important virus affecting lettuce in Hawaii, and we have transformed lettuce plants with the TSWV nucleocapsid (N) gene in various configurations. In some lines, the nucleocapsid protein is expected to be produced, and in others only the nucleocapsid RNA will be produced. Previous work has shown that resistance can be conferred by expressing viral RNA, and viral protein expression is not always required for resistance.
The lettuce plants that were directly transformed (the primary transformants) were allowed to set seed, and a PCR test confirmed that the majority of those progeny were transgenic. A small number of seeds from the primary transformants were produced, so in order to produce plants for virus challenge experiments, several of the progeny plants were allowed to set seed. This resulted in hundreds of seeds of those transgenic lines. These seeds are being propagated for segregation analysis, and for evaluation of virus resistance.
Developing orchids with resistance to cymbidium mosaic virus, and evaluating orchid virus detection kits:
The most important orchid viruses in Hawaii and worldwide are cymbidium mosaic virus (CymMV) and odontoglossum ringspot virus (ORSV). CymMV is more prevalent, but both viruses are serious challenges. We are working to develop transgenic orchids with resistance to CymMV, by transferring the CymMV coat protein gene to orchids. Our initial work is with Dendrobium varieties UH800 and UH306, which are very popular cut-flower varieties. We hope to provide virus resistant plants for Hawaiian cut-flower farms, which can have CymMV infection rates of over 95%.
We have cloned the CymMV coat protein (CP) gene and have developed vectors that will express this gene in both sense and antisense orientations. We are also in the process of transferring our constructs into a plant transformation vector for subsequent transfer to Agrobacterium. Protocorm-like bodies of the Dendrobium cultivars Jacquelyn Thomas UH800 and UH306 are being maintained in shaking culture, and these will be cocultivated with Agrobacterium to achieve gene transfer.
We are also using PCR as a sensitive virus detection method. We are doing this to resolve some serious discrepancies with the commercial antibody-based, orchid virus test kits. Some orchid growers use these kits, and these discrepancies are arising with recent large shipments of flasked orchids from overseas facilities.
The project is monitored via progress reports, meetings, site-visits, and telephone and email communications.